Patent application title: Novel reference plant, a method for its production, extracts obtained therefrom and their use

Abstract:

The invention relates to a reference plant which has been selected to:
a) not express a medicinally active compound or group of compounds; yet
b) express, at least substantially qualitatively, most other non
medicinally active compounds present in a therapeutically active
comparator plant.
The reference plant can be used to generate a reference extract with a
reference chemical profile which resembles that of the comparator plant
less the active compound or group of compounds and may thus be used as a
placebo or to otherwise test the hypothesis that the active compound or
compounds are responsible for an extracts perceived medicinal activity.

Claims:

1. A reference plant which has been selected to:a) not express a
medicinally active compound or group of compounds; yetb) express, at
least substantially qualitatively, most other non medicinally active
compounds present in a therapeutically active comparator plantsuch that
the reference plant can be used to generate a reference extract with a
reference chemical profile which resembles that of the comparator plant
less the active compound or group of compounds and may thus be used as a
placebo or to otherwise test the hypothesis that the active compound or
compounds are responsible for an extracts perceived medicinal activity.

2. A reference plant as claimed in claim 1 which is a cannabis plant and
the active compound or group of compounds are the cannabinoids.

3. A reference plant as claimed in claim 2 which contains a monogenic
mutation that blocks the cannabinoid biosynthesis in Cannabis sativa.

4. A reference plant as claimed in claim 2 wherein the reference plant
comprises a cannabinoid knock out factor governing a reaction in the
pathways towards the phenolic moieties olivetolic and divarinic acid.

5. A reference plant as claimed in claim 1 characterised in that a
homogenised bulk extract exhibits a profile of entourage compounds which
is quantitatively substantially similar to that of a reference plant.

6. A reference plant as claimed in claim 5 wherein the homogenised bulk
extract has a % v/w oil yield of greater than 0.14%, more preferably
greater than 0.2%, through 0.3% to 0.4% or more.

7. A reference plant as claimed in claim 1 characterised in that a
homogenised bulk steam distilled extract comprises both monoterpenes and
sesquiterpine.

18. A reference plant as claimed in claim 17 in which some trichomes
comprise headless; pinhead and/or shrivelled trichomes which may be flat,
convex or concave.

19. A reference plant as claimed in claim 15 in which the trichomes are
free of white trichome heads.

20. A reference plant as claimed in claim 1 characterized in that it
expresses monoterpenes, diterpenes, carotenoids, phytol and
tetraterpenes.

21. A reference plant as claimed in claim 1 characterized in that it
expresses sesquiterpenes, sterols and triterpenes.

22. A reference plant as claimed in claim 20 which expresses entourage
compounds selected from one or more of:monoterpenes; sesquiterpenes; and
flavonoids.

23. A reference plant as claimed in claim 1 having branching
characteristic of a drug producing phenotype as opposed to a fibre
producing phenotype.

24. A reference plant as claimed in claim 1 exhibiting vigour,
characterized in that the total above ground dry weight is substantially
equivalent to that of comparator drug producing plants.

25. A method of producing a reference plant which does not express a
medicinally active compound or group of compounds yet express, at least
substantially qualitatively, most other non medicinally active compounds
present in a therapeutically active comparator plant comprising:a)
Selecting a plant which does not express a medicinally active compound or
group of compounds;b) Selecting a therapeutically active comparator
plant; andc) Crossing the plant which does not express a medicinally
active compound or group of compounds with the therapeutically active
comparator plant to obtain an F1 progeny and self crossing the F1 progeny
to obtain an F2 progeny which is selected for the characteristics sought.

26. A method as claimed in claim 25 further comprising successive back
crosses with a comparator plant to selectively breed for the desired
characteristics.

27. An extract obtainable from a reference plant as claimed in claim 1.

28. A placebo comprising an extract as claimed in claim 27.

29. A method of testing a hypothesis that one or more compounds present in
a plant extract are responsible or are solely responsible for the
extracts pharmacological activity comprising:i) selecting a plant as
claimed in claim 1;ii) obtaining an extract therefrom; andiii) running
comparative tests against the extract obtained from a comparator plant.

30. A method of producing a designer plant extract comprising the steps
of:i) selecting a plant extract as claimed in claim 27; andii) combining
the extract of (i) with one or more medicinally active components.

Description:

RELATED APPLICATION

[0001]This application claims the benefit under 35 U.S.C. § 119(e) of
U.S. provisional application 60/932,437, filed May 31, 2007, the entire
disclosure of which is incorporated herein by reference.

FIELD OF THE INVENTION

[0002]The present invention relates to a novel reference plant, a method
for producing a novel reference plant, extracts free of a medicinally
active compound or group of compounds obtained therefrom and their use.
More particularly, the novel reference plant is a plant derived from a
comparator plant. In an exemplifying embodiment the medicinal compounds,
which are "knocked out", are one or more cannabinoids and the plant is
cannabis, Cannabis sativa, plant.

BACKGROUND OF THE INVENTION

[0003]Many pharmaceuticals are derived from plants and indeed many plants
or extracts obtained therefrom are taken as medicines. There are over 120
distinct chemical substances derived from plants that are considered as
important drugs that are currently in use. The table below lists some of
these substances.

[0004]There are many examples of plant-based substances that are known for
their medicinal properties. For example a tropical plant, Cephaelis
ipecacuanha, is known to produce the chemical emetine. A drug was
developed from this substance called Ipecac; this was used for many years
to induce vomiting. Another example of plant-based substances used as
medicines is the plant chemical named taxol found in the Pacific Yew
tree. The taxol molecule was produced synthetically to produce the drug
PACLITAXEL®, which is used in the treatment of various types of
tumours.

[0005]The plant substance, cynarin, is a plant chemical found in the
common artichoke (Cynara scolymus). A cynarin drug is sold for the
treatment of liver problems and hypertension. The drug is simply an
extract from the artichoke plant that has been standardized to contain a
specific amount of cyanarin. Similarly the substance silymarin is a
chemical found in the milk thistle plant and natural milk thistle
extracts that have been standardized to contain specific amounts of
silymarin are also used for the treatment of liver problems.

[0006]Some of the drugs/chemicals shown in the table below are sold as
plant based drugs produced from processing the plant material. Many plant
chemicals cannot be completely synthesised in the laboratory due to the
complex nature of the plant extract. For example the tree Cinchona
ledgeriana produces the substance quinine, which is used in to treat and
prevent malaria. Quinine is now chemically synthesised; however, another
chemical in the tree called quinidine, which was found to be useful for
the treatment of heart conditions, couldn't be completely copied in the
laboratory. The tree bark is used to produce a quinidine extract.

[0007]The table below details some of the plant-based medicines that are
in use today.

[0008]There are many examples of extracts that are characterized by
reference to a supposed active or marker. The principle described herein
with reference to cannabis plants would thus be applicable to other plant
types as are shown in the table above.

[0009]As an example of a botanical drug, Cannabis sativa has been used as
a drug for centuries, although the precise basis for the plants activity
is not known. Both THC and CBD, two of the plants cannabinoids, are known
to have distinct pharmacological activities and Marinol® (THC) and
Sativex® (an extract containing defined amounts of both THC and CBD)
are approved products for various medical indications.

[0010]In the case of extracts it is of course unclear whether the efficacy
of a botanical drug extract is attributable to the identified "active(s)"
or "markers" and/or other components present in an extract which may
provide an unidentified additive or synergistic effect or in fact be
directly responsible for the activity.

[0011]In the case of cannabis the supposed actives, the cannabinoids, are
produced through a series of enzymatic synthesis which are outlined
below:

[0012]The first specific step in the pentyl cannabinoid biosynthesis is
the condensation of a terpenoid moiety, geranylpyrophosphate (GPP), with
the phenolic moiety, olivetolic acid (OA; 5-pentyl resorcinolic acid), to
form cannabigerol (CBG). This reaction is catalysed by the enzyme
geranylpyrophosphate:olivetolate geranyltransferase (GOT); [1].
Precursors for GPP are the C5 isomers isopentenyl pyrophosphate
(IPP) and dimethylallyl pyrophosphate (DMAPP). These compounds can
originate from two different pathways: [0013]the mevalonate pathway
(MVA) that is located in the cytoplasm; and [0014]the deoxyxylulose
pathway (DOX) that operates in the plastid compartments.

[0015]According to Fellermeier et al. [2], the GPP incorporated into
cannabinoids is derived predominantly, and probably entirely, via the DOX
pathway of the glandular trichome plastids. The phenolic moiety OA is
generated by a polyketide-type mechanism. Rahaijo et al. [3] suggest that
n-hexanoyl-CoA and three molecules of malonyl-CoA condense to a C12
polyketide, which is subsequently converted into OA by a polyketide
synthase.

[0016]CBG is the direct precursor for each of the compounds THC [4], CBD
[5] and CBC [6], [7] and [8]. The different conversions of CBG are
enzymatically catalysed, and for each reaction an enzyme has been
identified: THC acid synthase [4] CBD acid synthase [5] and CBC acid
synthase [7] and [8].

[0017]Cannabinoids with propyl side chains, as identified by Vree et al.
[9] and de Zeeuw et al. [10], result if GPP condenses with divarinic acid
(DA; 5-propyl resorcinolic acid) instead of OA, into cannabigerovarin
(CBGV). The condensation of n-hexanoyl-CoA and two, instead of three,
molecules of malonyl-CoA, results in a C10 polyketide, which is
subsequently cyclisised into DA by a polyketide [11]. The three
cannabinoid synthase enzymes are not selective for the length of the
alkyl side chain and convert CBGV into the propyl homologues of CBD, THC
and CBC, which are indicated as cannabidivarin (CBDV), delta
9-tetrahydrocannabivarin (THCV) and cannabichromevarin (CBCV),
respectively [12].

SUMMARY OF THE INVENTION

[0018]The above pathway information is provided, as it will assist in an
understanding of the probable mechanism--giving rise to the zero
cannabinoid plants exemplifying the broader aspects of the invention.

[0019]Indeed it would be particularly useful to develop "knock out" plants
in which the one or more "actives" or "markers" believed to be
characteristic of a plants pharmaceutical activity are not expressed.
Such plants would be useful in formulating "true" placebo extracts or
comparator extracts for clinical trials and for producing extracts which
could be used in pharmacological tests and experiments in order that a
better understanding of an extract, and its perceived actives/markers
activity.

[0020]In the case of cannabis, the plant produces a vast array of
cannabinoids (including THC and CBD--the main perceived cannabinoid
actives) as well as a number of `entourage` compounds. Entourage
compounds are compounds which are related to cannabinoids but have little
or no activity at the cannabinoid receptors. Such entourage compounds are
thought to behave as modifiers of cannabinoid activity and therefore
could enhance pharmacological efficacy. It would be useful to have a
plant which did not produce the cannabinoids BUT which produced the
entourage compounds and other significant compounds in
combinations/amounts which at least substantially qualitatively and
preferably also substantially quantitatively resembled that of a
comparator plant, i.e. one which chemotypically bears a recognizable
resemblance to the medicinal plants used to generate a pharmaceutical or
medicine or a nutraceutical or functional food.

[0021]According to a first aspect of the present invention there is
provided a reference plant which has been selected to: [0022]a. not
express a medicinally active compound or group of compounds; yet express,
at least substantially qualitatively, most other non medicinally active
compounds present in a therapeutically active comparator plant [0023]such
that the reference plant can be used to generate a reference extract with
a reference chemical profile which resembles that of the comparator plant
less the active compound or group of compounds and may thus be used as a
placebo or to otherwise test the hypothesis that the active compound or
compounds are responsible for an extracts perceived medicinal activity.

[0024]The term "most other" is taken herein to refer to an amount of non
medicinally active compounds expressed by the reference plant which is at
least greater than 50% (w/w) of the total compounds in the plant. In
specific embodiments the amount is greater than 60% (w/w) non medicinally
active compounds, or the amount is greater than 70% (w/w) non medicinally
active compounds, or the amount is greater than 80% (w/w) non medicinally
active compounds, or the amount is greater than 90% (w/w) non medicinally
active compounds, or the amount is greater than 95% (w/w) non medicinally
active compounds.

[0025]Preferably the reference plant is a cannabis plant and the active
compound or group of compounds are the cannabinoids.

[0026]The cannabis plant is preferably a Cannabis sativa plant containing
a monogenic mutation that blocks the cannabinoid biosynthesis. Preferably
the plant comprises a cannabinoid knock out factor governing a reaction
in the pathways towards the phenolic moieties olivetolic and divarinic
acid.

[0027]Significantly, the reference plant is characterised in that a
homogenised bulk extract exhibits a profile of entourage compounds, which
is quantitatively substantially similar to that of a reference plant; as
for example is shown in FIG. 3.

[0028]In one embodiment the homogenised bulk extract has a % v/w oil yield
of greater than 0.14%, more preferably greater than 0.2%, through 0.3% to
0.4% or more.

[0029]A homogenised bulk steam distilled extract comprises both
monoterpenes and sesquiterpines. The monoterpenes comprise detectable
amounts of at least myrcene, alpha pinene and beta pinene. Preferably the
combined myrcene, alpha pinene and beta pinenes comprise at least 50%,
through 60% to at least 70% of the monoterpenes detected. Preferably it
will also comprise one or more of limonine and optionally linalool and
cis- and/or trans-verbenol.

[0030]The sesquiterpenes preferably comprise at least carophyllene and
humulene and may further comprise carophyllene oxide.

[0031]Preferably humelene epoxide II is not detected in the reference
plant.

[0032]The reference plants of the invention preferably comprise stalked
glandular trichomes. These are present at a density comparable to those
present in comparator drug type cannabinoid producing plants. The
reference plants typically have small, grey, dull trichomes of various
shapes (FIG. 2a). Some trichomes comprise headless; pinhead and/or
shrivelled trichomes, which may be, flat, convex or concave. They are
also free of white trichome heads.

[0033]The reference plant may be further characterized in that it
expresses monoterpenes, diterpenes, carotenoids, phytol and
tetraterpenes. It additionally expresses sesquiterpenes, sterols and
triterpenes.

[0034]The reference plant is further characterized in that it exhibits
branching characteristic of a drug producing phenotype as opposed to a
fibre producing phenotype and vigour, characterized in that the total
above ground dry weight is comparable to drug producing phenotypes.

[0035]According to a further aspect of the present invention there is
provided a method of producing a reference plant which does not express a
medicinally active compound or group of compounds yet express, at least
substantially qualitatively, most other non medicinally active compounds
present in a therapeutically active comparator plant comprising:
[0036]a) Selecting a plant which does not express a medicinally active
compound or group of compounds; [0037]b) Selecting a therapeutically
active comparator plant; and [0038]c) Crossing the plant which does not
express a medicinally active compound or group of compounds with the
therapeutically active comparator plant to obtain an F1 progeny and
self-crossing the F1 progeny to obtain an F2 progeny which is selected
for the characteristics sought.

[0039]According to yet a further aspect of the present invention there is
provided an extract obtainable from a reference plant of the invention.

[0040]According to yet a further aspect of the present invention there is
provided an extract obtainable from a reference plant of the invention.
Such extracts may be prepared by any method generally known in the art,
for example by maceration, percolation, vaporisation, chromatography,
distillation, recrystallisation and extraction with solvents such as C1
to C5 alcohols (ethanol), Norflurane (HFA134a), HFA227 and supercritical
or subcritical liquid carbon dioxide. In particular embodiments the
extracts may, for example, be obtained by the methods and processes
described in International patent application numbers WO02/089945 and WO
2004/016277, the contents of which are incorporated herein in their
entirety by reference.

[0041]In one embodiment the extract is used or formulated as a placebo. In
particular embodiments such formulations and/or placebos may, for
example, be formulated as described International patent application
numbers WO01/66089, WO02/064109, WO03/037306 and WO04/016246, the
contents of which are incorporated herein in their entirety by reference.

[0042]According to a further aspect of the present invention there is
provided a method of testing a hypothesis that one or more compounds
present in a plant extract are responsible or are solely responsible for
the extracts pharmacological activity comprising: [0043]i) selecting a
plant according to the first aspect of the invention; [0044]ii) obtaining
an extract therefrom; and [0045]iii) running comparative tests against
the extract obtained from a comparator plant.

[0046]According to a further aspect of the present invention there is
provided a method of producing a designer plant extract comprising the
steps of: [0047]i) selecting an extract obtainable from a reference
plant according to the first aspect of the invention and [0048]ii)
combining the extract of (i) with one or more medicinally active
components.

[0049]By "designer plant extract" is meant a plant extract which includes
one or more medicinally active components which do not naturally occur in
the reference plant of part i).

[0050]In specific embodiments, the medicinally active components may be
purified naturally occurring compounds, synthetic compounds or a
combination thereof. In a specific embodiment the medicinally active
components may be present in a plant extract. This plant extract may be
an extract from a "drug producing" plant of the same species as the
reference plant of part i). Typically this drug producing plant will not
be the comparator plant to the reference plant of part i).

[0051]The invention is further described, by way of example only, with
reference to novel Cannabis sativa plants (and not specific varieties),
which do not express cannabinoids but which otherwise, resemble,
chemotypically, medicinal cannabis plants.

BRIEF DESCRIPTION OF THE DRAWINGS

[0052]The invention will be further described, by way of example only to
the following figures in which:

[0053]FIGS. 1a-d are GC chromatograms from different chemotype segregants
from a 2005.45.13 F2. progeny (Table 2) [0054]a: is from
cannabinoid-free plants; [0055]b: is from low content and THC predominant
plants; [0056]c: is from high content and THC predominant plants; and
[0057]d: is from high content and CBG predominant plants.The peaks at
8.2, 16.0 and 16.7 min. represent the internal standard, THC and CBG,
respectively;

[0058]FIG. 2a-d are microscopic images of the bracteole surfaces from
different chemotype segregants from the 2005.45.13 F2 progeny.
[0059]a: is from cannabinoid-free plants; [0060]b: is from low content
and THC predominant plants; [0061]c: is from high content and THC
predominant plants; and [0062]d: is from high content and CBG predominant
plants.(The bar represents 500 μm) and

[0067]By way of introduction it should be noted that there are many
different Cannabis sativa varieties and chemotypes. These include both
wild type plants and cultivated varieties. The cultivated varieties
include plants which have been cultivated as fibre producers (low THC
varieties); those that have been bred (illegally) for recreational use
(high THC) and more recently medicinal plants which have been selectively
bred for their cannabinoid content (one or more cannabinoids predominate)
and optionally the profile of e.g. entourage compounds.

[0068]In order to produce plants with the desired characteristics it was
necessary to "knock out" the expression of cannabinoids in a manner,
which did not detrimentally effect the production of e.g. entourage
compounds in the medicinal plants. How this was achieved is set out
below:

Identification of a Cannabinoid-Free Chemotype Plant.

[0069]Because in many countries cannabis cultivation is restricted to
fibre hemp cultivars having specified "low" levels (typically below
either 0.1 or 0.3% w/w of the dry floral tissue) of THC, several breeding
programmes have been devoted to meeting these legal limits.

[0070]According to a survey of the European commercial fibre cultivars
[13], the cultivars bred at the Ukrainian Institute of Fibre Crops
(Glukhov, formerly, Federal Research Institute of Bast Crops) have the
lowest THC contents and the lowest total cannabinoid contents. The
cannabinoid breeding programme at this institute started in 1973. Their
usual selective breeding methodology consists of family selection within
existing cultivars with a high agronomic value and the elimination,
before flowering, of plants with relatively high contents [14] and [15].
This effort has resulted in a gradual decrease of both THC content and
total cannabinoid content.

[0071]Gorshkova et al. [16] evaluated the densities of sessile and stalked
glandular trichomes on the bracteoles of various plants. They found that
plants with stalked trichomes had relatively high cannabinoid contents
and that their contents were positively correlated with the density of
the stalked trichomes. Plants that had solely sessile trichomes always
had low contents that were uncorrelated with the densities of the sessile
trichomes. Gorshkova et al. [16] also mention plants without glandular
trichomes that were found to be cannabinoid-free.

[0072]Since then, Ukrainian plant breeders have reported several times on
the existence of cannabinoid-free breeding materials [15], [17] and [18]

[0073]Pacifico et al. [1,9] analysed individual plants from the Ukrainian
cultivar USO 31 and found that one third of the individuals contained no
cannabinoids. He also found that a minority of the plants (<10%) in a
French fibre cultivar, Epsilon 68, were cannabinoid-free.

[0074]The Ukrainian cultivar USO 31 is amongst several varieties of hemp
that have been approved for commercial cultivation under subsection 39(1)
of the Industrial Hemp Regulations in Canada for the year 2007.

[0075]These cannabinoid free plants are phenotypically and chemotypically
different to those developed by the applicant through artificial
manipulation and differ from those cannabinoid free plants that have been
isolated in nature.

[0076]Theoretically, two different physiological conditions could make a
plant cannabinoid-free: [0077](1) a disrupted morphogenesis of
glandular trichomes that, according to Sirikantaramas et al. [20], appear
to be essential structures for cannabinoid synthesis, and [0078](2) a
blockage of one or more biochemical pathways that are crucial for the
formation of precursors upstream of CBG.

[0079]The first condition would also seriously affect the synthesis of
other secondary metabolites that are produced largely or uniquely in the
glandular trichomes.

[0080]In 1991, field grown cannabinoid-free plants, resulting from
Gorshkova et al. [16] programme were viewed and the bracts and bracteoles
of these plants were apparently lacking glandular trichomes. Also, the
plants did not exude the characteristic cannabis fragrance. This suggests
that the volatile mono- and sesquiterpenes were not produced in these
plants. Such cannabinoid free plants might therefore have been considered
unsuitable for the purpose of breeding a cannabinoid free plant with
typical entourage compounds.

[0081]The second condition could also affect metabolites other than
cannabinoids, as in the case of an obstruction of the basic pathways of
common precursors for different classes of end products.

[0082]The IPP incorporated, via GPP, into cannabinoids is derived from the
DOX pathway in the plastids [2]. Monoterpenes, diterpenes, carotenoids,
phytol and tetraterpenes are also uniquely synthesised in the plastids
and one could therefore conclude that the IPP incorporated in these
compounds, as with cannabinoids, is derived from the DOX pathway [21].

[0083]Sesquiterpenes, sterols and triterpenes are uniquely synthesised in
the cytoplasm. Presumably they are synthesised from MVA derived IPP [21]
and so do not share a fundamental pathway with the terpenoid moiety of
cannabinoids.

[0084]Even so, according to Evans [22], there is also evidence for a
cooperative involvement of the DOX- and the MVA pathway in the synthesis
of certain compounds, through the migration of IPP from the plastids into
the cytoplasm and vice versa.

[0085]The potentially wider chemical effect of engineering plants with the
cannabinoid knockout factor yet which express selected entourage
compounds has implications for pharmaceutical cannabis breeding.
Cannabinoids, and THC in particular, are generally considered as the
major pharmaceutically active components of Cannabis. Nevertheless,
according to McPartland and Russo [23], the terpenoid fraction may modify
or enhance the physiological effects of the cannabinoids, providing
greater medicinal benefits than the pure cannabinoid compounds alone. As
summarized by Williamson and Whalley [24], there are indications that the
non-cannabinoid `entourage` of constituents, such as:
[0086]monoterpenes; [0087]sesquiterpenes; and [0088]flavonoidsmodulate
the cannabinoid effects and also have medicinal effects by themselves.
Speroni et al. [25] reported an anti-inflammatory effect from an extract
that was obtained from a cannabinoid-free chemotype.

Selection

[0089]Whilst USO-31 was selected as the source of a "knockout" gene to be
introduced into pharmaceutical plants the challenge remained of achieving
plants which were devoid of cannabinoids but which retained a good
profile of selected entourage compounds (i.e. were broadly speaking
comparable to plants grown to produce extracts for pharmaceutical use).
In this regard USO-31 had a chemical profile, which was not similar to
medicinal varieties in that it was lacking both in cannabinoids, and
monoterpenes. Furthermore, the sesquiterpene profile also differed both
quantitatively and qualitatively from that of plants used to produce
pharmaceutical extracts.

EXAMPLES

Example 1

Breeding Programme

[0090]To overcome the problem of creating a reference plant which is, in
the case of Cannabis sativa, free of cannabinoids BUT which had a
chemical profile of entourage compounds resembling pharmaceutical
cannabis, selective breeding programmes were undertaken.

[0091]A first cross was made between the selected cannabinoid free plant
USO-31 and a plant having a high cannabinoid content of a given
cannabinoid, in this case M35, a high THCV containing plant (83.4% by
weight of cannabinoids THCV), and M84, a high CBD containing plant (92.4%
by weight of cannabinoids CBD). The high cannabinoid plants were selected
both for their high and specific cannabinoid contents and their vigour.

[0092]Alternatively, a direct cross with a selected pharmaceutical plant
could have been made.

[0094]Of course other strains containing a high percentage of another
cannabinoids e.g. THC, CBDV, CBG, CBGV, CBC, CBCV, CBN and CBNV could be
used. By "high" is meant that the specific cannabinoid predominates and
would typically comprise greater than 50% by weight of the total
cannabinoids present, more particularly greater than 60%, through 70% and
80% to most preferably greater than 90% by weight.

[0095]The initial cross generated an F1 progeny (Table 2 rows 1 and 2)
which were then self crossed to generate an F2 progeny from which plants
having the desired characteristics (zero cannabinoid/good entourage
compound chemotype profile) were selected for back crossing to
pharmaceutical varieties.

[0096]The selected zero-cannabinoid plant, USO-31, was monoecious. i.e. it
has unisexual reproductive units (flowers, conifer cones, or functionally
equivalent structures) of both sexes appearing on the same plant. In
order to self-fertilise USO-31 and mutually cross female plants, a
partial masculinisation was chemically induced. Self-fertilisations were
performed by isolating plants in paper bags throughout the generative
stage. The USO-31 source plants were evaluated for their drug type habit.
Inbred seeds from the best individual apparently devoid of cannabinoids
and another with only cannabinoid traces were pooled.

[0098]Similarly, the 19 plants of the 2003.17 F1 comprised a majority
of individuals with a cannabinoid content in the range of from 1.69 to
13.76%, and two plants with cannabinoid traces of only ca. 0.02%.

[0099]From both F1s, an individual with only trace cannabinoid
amounts was self-fertilised to produce an inbred F2 2003.8.21 and
2003.17.19. Both F2s comprised plants that were confirmed to be
devoid of cannabinoids.

[0100]The remaining plants, those with cannabinoids present, could be
assigned to two categories on the basis of a discontinuity in the
cannabinoid content range: [0101]a group with low contents ranging from
trace amounts up to roughly 0.6%; and [0102]a group with higher contents.

[0103]The newly obtained cannabinoid-free plants designated 2003.8.21 and
2003.17.19 F2 had more branching (typical of a drug type phenotype
and in contrast to that of a fibre type phenotype), a stronger fragrance
(due to the presence/increase in the terpenes and sesquiterpenes) and
higher trichome density (determinable on examination) than the original
USO-31 plants.

[0105]Backcrosses were performed in order to obtain cannabinoid-free
material, more closely resembling (both qualitatively and quantitatively)
the pharmaceutical production clones by way of their non-cannabinoid
profile, particularly those of the entourage compounds.

[0106]All the Clones Listed in Table 1 were True Breeding for their
Chemotype.

ii) Backcrossing of Cannabinoid-Free Lines to Pharmaceutical Production
Clones M3 and M16

[0107]The cannabinoid-free lines 2003.8.21.76 and 2003.17.19.67, (Table 2,
column 2, last 4 rows) were then back crossed with pharmaceutical
production clones M3 and M16 and the resulting F1's crossed to generate
an F2 progeny.

[0109]Within the F1s the cannabinoid contents showed a single
Gaussian distribution. The F1 contents were much lower than the
parental means and therefore much closer to the cannabinoid-free parent
than to the production parent. The F1s were well covered with
trichomes and were quite fragrant.

[0110]In respect of the cannabinoid composition, the 2005.45 F1
segregated into two chemotypes: THC predominant plants and mixed CBD/THC
plants, in a 1:1 ratio.

[0111]The 2005.46 F1 had a uniform CBD chemotype.

[0112]The 2005.47 F1 was uniform and consisted of THC plants, all
with a minor proportion of THCV.

[0113]The 2005.48 F1 was uniform and consisted of CBD/THC plants that
also had minor proportions of CBDV and THCV.

[0114]Per F1, one individual was selected on the basis of criteria
such as `drug type morphology` (e.g. branching) and minimal
monoeciousness to produce back cross generations. These individuals were
used for a repeated pollination of M3 or M16, which is not discussed
here.

[0115]To examine chemotype segregation, the selected F1 individuals
were also self-fertilised to produce large inbred F2s.

[0116]FIG. 1 shows chromatograms of different chemotype segregants from
the 2005.45.13 F2. FIG. 1a is the chromatogram for a zero
cannabinoid plant.

[0117]The different chemotype segregants were microscopically compared.
The cannabinoid-free plants of each progeny all had small, grey, dull
trichomes of various shapes (FIG. 2a). Some were headless; some were
pinhead and shrivelled, either flat, convex or concave.

[0118]By way of contrast:

[0119]The high content CBD- and/or THC-predominant individuals of each
group all had big, round clear heads that sparkled under the lamp (FIG.
2b);

[0120]The low content plants from each progeny were almost
indistinguishable from the cannabinoid-free plants except that there was
an occasional small but bright trichome in some (FIG. 2c); and

[0121]The high content CBG predominant plants from the 2005.45.13 F2
had big, round, opaque white heads (FIG. 2d), clearly distinct from the
transparent ones occurring on the THC predominant plants of the same
progeny.

[0122]The low content CBG predominant 2005.45.13 plants did not show
opaque white trichome heads and were indistinguishable from the low
content THC predominant plants. Neither were white trichome heads
observed in any of the cannabinoid-free plants of this progeny.

[0123]As an indication of their vigour, the total above ground dry weights
of all the cannabinoid-free- and the high content segregants were
assessed. Per progeny, per segregant group the weights showed a Gaussian
distribution.

[0124]For the 2005.45.13, 2005.46.27 and the 2005.47.9 progenies the
cannabinoid-free individuals on average had a ca. 10% higher dry weight
than the high content individuals.

[0125]In the 2005.48.7 progeny however, the average weight of the high
content group exceeded that of the cannabinoid-free group by about 10%.

[0126]In order to characterize the plants a chemical analysis of both the
cannabinoid content, and selected other chemicals, was undertaken as set
out below:

Example 2

i) Analysis of Cannabinoid Content and Other Chemicals

[0127]Mature floral clusters were sampled from every individual plant
considered in the breeding experiments. Sample extraction and GC analysis
took place as described by de Meijer et al. [26].

[0128]The identities of the detected compounds were confirmed by GC-MS.
Cannabinoid peak areas were converted into dry weight concentrations
using a linear calibration equation obtained with a CBD standard range.
The contents of the individual cannabinoids were expressed as weight
percentages of the dry sample tissue. The total cannabinoid content was
calculated and the weight proportions of the individual cannabinoids in
the cannabinoid fraction were used to characterize the cannabinoid
composition.

ii) Chemical Comparison of Bulk Segregants

[0129]Each of the six F2s listed in Table 2 segregated into:
[0130]cannabinoid-free plants; [0131]plants with cannabinoid traces; and
[0132]plants with high cannabinoid contents.

[0133]In each case, per F2, the floral leaves, bracts and bracteoles
of all the cannabinoid-free plants were pooled and homogenised, as was
the floral fraction of all the plants belonging to the group with high
cannabinoid contents. The different bulks from the: [0134]2005.45.13
(from M3-THC), [0135]2005.46.27 (from M16-CBD), [0136]2005.47.9 (from
M3-THC) and [0137]2005.48.7 (from M16-CBD) F2 were steam-distilled
and the essential oil yields were assessed.

[0138]The monoterpene and sesquiterpene composition of these essential
oils was analysed by Gas Chromatography with Flame Ionisation Detection
(GC-FID).

[0139]The relative amounts of a wide range of entourage compounds in the
bulk homogenates of: [0140]2003.8.21 (from M84-CBD) and
[0141]2003.17.19 (from M35 THCV) F2swere also compared by using the
following analytical techniques:

a) Gas Chromatography--Mass Spectrometry (GC-MS)

[0142]To obtain comparative fingerprints, GC-MS analyses were performed on
a HP5890 gas chromatograph, coupled to a VG Trio mass spectrometer. The
GC was fitted with a Zebron fused silica capillary column (30
m×0.32 mm inner diameter) coated with ZB-5 at a film thickness of
0.25 μm (Phenomenex). The oven temperature was programmed from
70° C. to 305° C. at a rate of 5° C./min. Helium was
used as the carrier gas at a pressure of 55 kPa. The injection split
ratio was 5:1.

b) Gas Chromatography with Flame Ionisation Detection (GC-FID)

[0143]GC profiles of terpenoids were generated in the splitless mode with
a HP5890 gas chromatograph. The GC was fitted with a Zebron fused silica
capillary column (30 m×0.32 mm inner diameter) coated with ZB-624
at a film thickness of 0.25 μm (Phenomenex). The oven temperature was
held at 40° C. for 5 minutes, programmed to 250° C. at a
rate of 10° C./min then held at 250° C. for 40 minutes.
Helium was used as the carrier gas at a pressure of 9.2 psi. The
injection split ratio was 10:1.

[0144]HPLC profiles were obtained using methods specific to a variety of
compound classes. All samples were analysed using Agilent 1100 series
HPLC systems

(i) Cannabinoid profiles were generated using a C18 (150×4.6
mm, 5 μm) analytical column. The mobile phase consisted of
acetonitrile, 0.25% w/v acetic acid and methanol at a flow rate of 1.0
ml/min and UV profiles were recorded at 220 nm.(ii) Carotenoid profiles
were generated using a Varian Polaris C18 (250×4.6 mm, 5
μm) analytical column. The mobile phase consisted of
acetonitrile:methanol:dichloromethane: water at a flow rate of 1.2 ml/min
and UV profiles were recorded at 453 nm.(iii) Chlorophyll profiles were
generated using the same column, mobile phase and flow rate described for
carotenoids. UV profiles were recorded at 660 nm.(iv) Non-polar compound
profiles (triglycerides, sterols etc) were generated by a gradient LC
method using a Phenomenex Luna C18 (2) (150×2.0 mm, 5 μm)
analytical column. The mobile phase consisted of solvent A
(acetonitrile:Methyl-tert-butyl-ether (9:1)) and solvent B (water) with
the proportion of B decreased linearly from 13% to 0% over 30 minutes
then held constant for 20 minutes at a flow rate of 1.0 ml/min. The flow
rate was then increased linearly to 1.5 ml/min over 40 minutes and UV
profiles were recorded at 215 nm.

[0145](v) Polar compound profiles (phenolics) were generated by a gradient
LC method using an Ace C18 (150×4.6 mm, 5 μm) analytical
column. The mobile phase consisted of solvent A (acetonitrile:methanol,
95:5) and solvent B (0.25% w/v acetic acid:methanol, 95:5). The
proportion of B was decreased linearly from 75% to 15% over 30 minutes
then held constant for 10 minutes at a flow rate of 1.0 ml/min and UV
profiles were recorded at 285 nm.

Results

Chemical Comparison of Cannabinoid-Free- and High Content Bulks

[0146]The yields and compositions of steam-distilled essential oils from
bulked cannabinoid-free- and bulked high content segregants of the four
F2 progenies are presented in Table 4 below.

[0147]In three (2005.46.27, 2005.47.9, and 2005.48.7), the
cannabinoid-free bulks contained less essential oil than the high content
ones.

[0148]In 2005.45.13 however, the cannabinoid-free bulk was slightly
richer.

[0149]No significant qualitative differences in the essential oil
composition were found, only minor quantitative ones, which generally did
not show a systematic pattern.

[0150]The only consistent quantitative difference between the low and high
content progeny was difference was found for caryophyllene oxide that in
all four progenies, reached a higher proportion in the cannabinoid-free
bulks than in the high content bulks.

[0151]When the zero cannabinoid backcross plants of the invention were
compared to control 1 (the original zero cannabinoid plant which was also
devoid of monoterpenes) and controls 2 and 3 (the pharmaceutical plants
with a high cannabinoid content and a range of entourage compounds) the
following differences were observed: [0152]1. The volume of oil (%)
obtained by steam distillation in the zero cannabinoid plants of the
invention was on average 0.50%. By way of comparison control 1 is 0.14%,
and the mean of control 2 and 3 was 0.52%. In other words the % oil is
representative of the pharmaceutical clones. [0153]2. The total measured
monoterpene fraction in the zero cannabinoid plants of the invention was
on average about 76. By way of comparison control 1 is 0, and the mean of
control 2 and 3 was about 61. In other words the monoterpene fraction is
representative of the pharmaceutical clones. [0154]3. Within the
monoterpence fraction in the zero cannabinoid plants of the invention the
predominant terpene was myrcene, followed by alpha pinine and beta pinine
with smaller amounts of limonine and linalol. Whilst quantitatively there
were differences compared to the pharmaceutical controls there was,
broadly speaking, a qualitative relationship. [0155]4. The total measured
sesquiterpene fraction in the zero cannabinoid plants of the invention
was on average about 23. By way of comparison, control 1 is about 93, and
the mean of controls 2 and 3 was about 39. In other words the
sesquiterpene fraction is much more representative of the pharmaceutical
clones than control 1. [0156]5. Within the sesquiterpene fraction in the
zero cannabinoid plants of the invention the predominant sesquiterpene
were carophyllene, humulene and carophyllene oxide (accounting for more
than 50% of the sesquiterpence fraction). Whilst there were differences
compared to the pharmaceutical controls (where quantitatively
carophyllene and humulene were again the most significant sesquiterpenes
but carophyllene oxide was absent) there was, broadly speaking a
qualitative, if not quantitative relationship between the plants of the
invention and the pharmaceutical plants as compared to the starting zero
cannabinoid plants which had much higher levels of sesquiterpenes and a
wider detectable range of sesquiterpenes.

[0157]By way of comparison Table 5 gives some analytical data on the
intermediate plants generated. It is a comparison of the different
segregant bulks from 2003.8.21 and 2003.17.19 for a variety of compound
classes.

[0158]In general the differences between the entourages of the
cannabinoid-free and the high content bulks were only quantitative.
Limonene was an exception, as it was not detected in the cannabinoid-free
bulks whereas a minor presence was found in both of the high content
bulks.

[0159]However, the essential oil data in Table 4 does not confirm this
finding for the other F2s. Likewise, Table 5 does not show the
difference in caryophyllene oxide as it appears in Table 4.

[0160]Both progenies in Table 5 had consistently higher levels of four
different triglycerides in the cannabinoid-free bulks than the high
content bulks. The occurrence of none of the entourage compounds listed
in the Tables 4 and 5 appears to be critically associated with the
presence or absence of cannabinoids.

[0161]With the reported exception of the triglycerides, the quantitative
differences in the entourage compounds does not show a consistent trend
between cannabinoid-free- and high content bulks.

[0162]This is most clearly seen in FIG. 3, which compares high cannabinoid
bulks with cannabinoid free bulks. It also shows an M3 pharmaceutical
bulk. What is apparent from a comparison of these extracts is that the
profiles between the high content bulk and the cannabinoid free bulk of
the segregating plants are very similar and that further more there is
substantial similarity to the pharmaceutical extract M3, particularly at
the earlier retention times (less than 30 minutes).

Discussion

[0163]The cannabinoid-free segregants resulting from backcrosses with high
content drug clones had glandular trichomes in normal densities but the
trichome heads were dull and much smaller than those of high cannabinoid
content sister plants. Nevertheless the trichomes of cannabinoid-free
segregants appear to be functional metabolic organs, as the chemical
comparison of contrasting segregant bulks did not reveal big differences
in the content and composition of volatile terpenes, which are also
produced in the trichomes. The absence of cannabinoids probably causes
the small trichome heads, rather than being a result of them.

[0164]The abundant presence of apparently functional trichomes on the
cannabinoid-free plants rules out that the absence of cannabinoids is due
to a disrupted morphogenesis of the glandular trichomes. It thus appears
that the cannabinoid knockout factor is not derived from the gland free
plants selected by Gorshkova et al [16].

[0165]It is more plausible that the absence of cannabinoids is
attributable to the blockage of one or more biochemical pathways that are
crucial for the formation of precursors upstream of CBG. As the chemical
entourage of cannabinoid-free plants is intact, the obstacle is probably
not in the MVA and DOX pathways towards IPP.

[0166]A blocked MVA pathway would not affect cannabinoid synthesis [2],
but it should reduce levels of sesquiterpenes, sterols and triterpenes
[21].

[0167]A blockage of the DOX pathway would obstruct the synthesis of the
terpenoid moiety of cannabinoids [2] but it should also negatively affect
the synthesis of monoterpenes, diterpenes, carotenoids, phytol and
tetraterpenes [21].

[0168]An alternative is that the knockout allele encodes a defective form
of the enzyme GOT [1] that catalyses the condensation of resorcinolic
acids (OA and DA) with GPP into CBG. However, with such a mechanism one
would expect an accumulation of the phenolic moieties OA and/or DA in the
cannabinoid-free segregants. Our GC method for cannabinoid analysis
detects the decarboxylated forms of both acids but they were observed in
none of the cannabinoid-free plants' chromatograms.

[0169]The most plausible hypothesis for the absence of cannabinoids
appears to be a blockage in the polyketide pathway towards the phenolic
moieties OA and DA. Whatever the working mechanism of the cannabinoid
knockout factor is, one would expect that a functional synthase dominates
a non-functional version, and so it remains obscure as to why the
heterozygous genotypes (O/o) have such a strongly suppressed cannabinoid
synthesis.

[0170]The essential oil comparison and the chromatographic fingerprinting
of contrasting segregant bulks demonstrated that except the cannabinoids,
all the monitored compound classes were present in both segregant groups.
The relative levels of the compound classes did vary between the
contrasting segregant groups but not usually in a systematic way.

[0171]The quantitative differences between contrasting bulks could be
attributable to the fact that in cannabinoid-free plants the trichome
heads, as the metabolic centres for a range of end products, are not
inflated with cannabinoids. This may change the physical environment in
which the reactions occur so that it quantitatively affects the synthesis
of entourage compounds. The fact that large amounts of basic cannabinoid
precursors are not incorporated may also affect equilibriums of other
biosynthetic reactions.

[0172]A further benefit of the plants of the present invention is that
they can be used to create plant extracts containing cannabinoids in
quantities/purities, which could not be achieved naturally. Such plant
extracts providing the benefits arising from the presence of one or more
selected entourage compounds. The cannabinoids, which could be introduced
to the cannabinoid free extracts, could include one or more natural
cannabinoids, synthetic cannabinoids or biosynthetic cannabinoids
(modified natural cannabinoids). This would produce a "designer" plant
extract that could be used in clinical trials or as medicines.

[0173]The benefits of natural or biosynthetic cannabinoids over synthetic
cannabinoids lies in the fact that all of the cannabinoids are in the
active form as opposed to a racemic mixture.

[0174]Other aspects of the invention will be clear to the skilled artisan
and need not be repeated here. Each reference cited herein is
incorporated by reference in its entirety for the relevant teaching
contained therein.

[0175]The terms and expressions that have been employed are used as terms
of description and not of limitation, and there is no intention in the
use of such terms and expressions of excluding any equivalents of the
features shown and described or portions thereof, it being recognized
that various modifications are possible within the scope of the
invention.